Nucleotide excision repair (NER) is an important cellular defense mechanism against mutagenesis and carcinogenesis. Defects in this repair pathway also lead to complex human hereditary diseases such as xeroderma pigmentosum, Cockayne syndrome, and trichothiodystrophy. Thus, understanding the biochemical process of NER and identify all of the components in the pathway will not only enable us to gain insights into a fundamental biological function, but also contribute to the understanding of human carcinogenesis, as well as human repair diseases. The objectives of this proposal are to define the biochemical pathway of NER in eukaryotes using the yeast S. cerevisiae as a model system and to understand the regulatory aspects of NER-transcription relation connected together by the transcription factor TFllH. The objectives will be achieved by identifying proteins required in DNA damage recognition/incision/excision, DNA repair synthesis, and DNA ligation, which constitute the NER pathway. The roles of Rad7, Radl6, and Rad23 in NER will be investigated. The relationship between NER and Kin28 or Ccl1, the kinase and cyclin subunits of yeast holo TFIIH, respectively, will also be examined. This proposal will utilize a combination of approaches including a yeast cell-free system for NER, protein and protein complex purification and characterization, functional assays, dissection of NER pathway into individual biochemical steps, and molecular cloning. When successfully carried out, these proposed studies should lead to the identification of all yeast proteins required in NER, thus, defining a complete biochemical pathway for the repair.